Recombinant Bacillus anthracis Potassium-transporting ATPase C chain (kdpC)

What is the basic structure and function of the KdpC protein in Bacillus anthracis?

The KdpC protein functions as a critical subunit of the high-affinity potassium-transporting ATPase complex in B. anthracis. It works in conjunction with KdpA, KdpB, and KdpF to form a complete ATP-driven K⁺ transport system. Structurally, KdpC serves as a stabilizing peripheral membrane component that interacts with the catalytic KdpB subunit. The Kdp system is particularly important when B. anthracis encounters potassium-limited environments, as might occur during host infection. Recent research indicates that potassium homeostasis is linked to virulence factor expression in B. anthracis, with c-di-AMP signaling serving as an important regulatory mechanism affecting potassium uptake systems .

What expression systems are most effective for producing recombinant B. anthracis KdpC?

For recombinant expression of B. anthracis KdpC, the E. coli BL21(DE3) strain has proven effective when coupled with pET-based expression vectors. The protein can be expressed with either N-terminal or C-terminal His-tags to facilitate purification. Optimal expression conditions typically involve induction with 0.5-1.0 mM IPTG at lower temperatures (16-25°C) for extended periods (12-18 hours) to enhance protein solubility. Since KdpC is normally associated with the membrane but is not itself a transmembrane protein, it can be expressed in the soluble fraction under appropriate conditions. Alternative expression systems including B. subtilis may also be considered for more native-like post-translational modifications, though yields are typically lower than with optimized E. coli systems.

What are the optimal purification strategies for recombinant B. anthracis KdpC?

The most effective purification strategy for recombinant B. anthracis KdpC involves a multi-step process:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins for His-tagged KdpC. The optimal buffer conditions include 50 mM Tris-HCl pH 8.0, 300 mM NaCl, with imidazole gradients (10-250 mM) for washing and elution.

• Intermediate purification: Ion exchange chromatography (typically anion exchange using Q-Sepharose) can separate KdpC from remaining contaminants.

• Polishing step: Size exclusion chromatography using Superdex 75 or 200 columns in 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol.

The addition of low concentrations of potassium (5-10 mM KCl) in all buffers can help maintain protein stability. Furthermore, the inclusion of reducing agents (1-5 mM DTT or 1-2 mM β-mercaptoethanol) prevents unwanted oxidation of cysteine residues. Typical yields range from 5-15 mg of purified protein per liter of bacterial culture, with purity exceeding 95% as assessed by SDS-PAGE and mass spectrometry.

How can researchers effectively assess the interaction between KdpC and other components of the Kdp system?

To assess interactions between KdpC and other Kdp system components, researchers should employ multiple complementary approaches:

• Co-immunoprecipitation: Using antibodies against KdpC or epitope tags to pull down interacting partners, followed by Western blot or mass spectrometry analysis.

• Bacterial two-hybrid assays: These can map specific interaction domains between KdpC and other Kdp components, particularly KdpB with which it forms a tight association.

• Surface plasmon resonance (SPR): This provides quantitative binding kinetics between purified KdpC and other Kdp proteins, with typical KD values in the nanomolar range for physiological interactions.

• Microscale thermophoresis (MST): Useful for detecting interactions in solution with minimal protein consumption.

• Native PAGE and crosslinking studies: These can capture transient interactions and complex formation.

For functional reconstitution, researchers have successfully employed proteoliposomes containing the complete KdpFABC complex to measure ATPase activity and potassium transport. This system requires careful optimization of lipid composition and protein-to-lipid ratios to maintain native-like activity.

How does c-di-AMP signaling modulate the expression and function of the Kdp system in B. anthracis?

c-di-AMP (cyclic di-adenosine monophosphate) regulates the Kdp system through multiple mechanisms, creating a sophisticated control network:

• Transcriptional regulation: c-di-AMP inhibits the expression of the Kdp operon through binding to the ydaO riboswitch, effectively reducing the synthesis of KdpFABC components including KdpC .

• Two-component system interference: c-di-AMP binds to the sensor kinase KdpD, modulating its autophosphorylation and subsequent phosphotransfer to KdpE, the response regulator that controls kdp operon expression .

• Functional implications: High intracellular c-di-AMP levels inhibit potassium uptake systems, correlating with decreased anthrax toxin expression in B. anthracis .

The intricate relationship is summarized in the following table:

Researchers investigating this relationship should consider employing c-di-AMP analogs and Kdp system mutants to delineate the precise molecular mechanisms underpinning this regulatory network. Phosphorylation status of KdpE can be monitored using Phos-tag gel electrophoresis or mass spectrometry approaches.

What structural features of KdpC contribute to its role in the assembled Kdp-ATPase complex?

KdpC contains several structural elements crucial for its function within the Kdp-ATPase complex:

• N-terminal domain: Contains a βαββαβ motif that interacts with the nucleotide-binding domain of KdpB.

• Central region: Features several conserved charged residues that stabilize the interaction with KdpB and potentially contribute to conformational changes during the transport cycle.

• C-terminal segment: Likely involved in fine-tuning the ATPase activity of the complex through allosteric mechanisms.

Analysis of KdpC homologs from related species reveals several highly conserved motifs, including a GxxxG-like sequence that may facilitate protein-protein interactions within the complex. Structure-function studies using site-directed mutagenesis have identified specific residues in KdpC that, when altered, affect ATPase activity or stability of the complex. Crystal structures of Kdp complexes from model organisms suggest that KdpC undergoes significant conformational changes during the catalytic cycle, highlighting its dynamic role in the transport mechanism.

How can researchers effectively study the relationship between potassium transport and virulence in B. anthracis?

Investigating the relationship between potassium transport and virulence in B. anthracis requires a multifaceted approach:

• Generation of targeted mutants: Creating kdpC knockout and conditional expression strains using CRISPR-Cas9 or allelic exchange methods specific for B. anthracis.

• Virulence factor expression analysis: Monitoring toxin component production (protective antigen, lethal factor, edema factor) in wild-type versus kdpC mutant strains under various potassium concentrations using Western blotting, ELISA, or reporter systems .

• Transcriptomics and proteomics: RNA-seq and mass spectrometry-based approaches to profile global changes in gene expression and protein abundance in response to potassium limitation or kdpC mutation.

• Infection models: Comparing virulence of wild-type and kdpC mutant strains in appropriate animal models or cell culture systems, with particular attention to patterns of bacterial dissemination, toxin production, and host survival rates.

• Potassium flux measurements: Employing potassium-selective electrodes or fluorescent indicators to measure real-time potassium transport in live bacteria under various conditions.

Researchers have observed that disruption of potassium homeostasis affects anthrax toxin expression, with evidence suggesting that increased intracellular potassium levels promote toxin expression in B. anthracis strains with accumulated c-di-AMP . This indicates a potential regulatory mechanism where potassium serves as a signal for virulence gene expression.

What are the main challenges in differentiating the roles of different potassium transport systems in B. anthracis?

B. anthracis possesses multiple potassium transport systems, including the high-affinity Kdp system and the lower-affinity Ktr system, presenting several research challenges:

• Functional redundancy: Multiple potassium transport systems can compensate for each other, making phenotypic analysis of single-system mutants difficult. This can be addressed by creating combinatorial mutants affecting multiple transport systems and analyzing their phenotypes under carefully controlled potassium concentrations.

• Environmental regulation: Different transport systems are activated under distinct conditions, necessitating careful experimental design. For example, the Kdp system is typically induced under severe potassium limitation (<0.1 mM K⁺), while the Ktr system functions under moderate potassium concentrations.

• Integration with other cellular processes: Potassium transport intersects with pH homeostasis, osmotic regulation, and membrane potential, complicating interpretation of experimental results. Researchers should employ membrane potential dyes, pH indicators, and osmolarity controls in their experimental setups.

To address these challenges, researchers have developed specific inhibitors of different potassium transport systems and employed radioactive ⁸⁶Rb⁺ as a tracer for potassium flux measurements with high specificity. Additionally, electrophysiological techniques such as patch-clamping and ion-selective microelectrodes can provide direct measurements of transport activity with high temporal resolution.

How can researchers ensure proper folding and assembly of recombinant KdpC for structural studies?

Ensuring proper folding and assembly of recombinant KdpC for structural studies presents several challenges that can be addressed through the following approaches:

• Co-expression strategies: Express KdpC alongside its natural binding partner KdpB to promote proper folding and complex formation. This can be achieved using polycistronic expression vectors or co-transformation of compatible plasmids.

• Protein stabilization: Include stabilizing agents in buffers such as glycerol (10-15%), specific lipids (particularly phosphatidylglycerol), and physiologically relevant concentrations of potassium (5-10 mM).

• Quality control methods:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Thermal shift assays to assess protein stability

  • Dynamic light scattering (DLS) to evaluate monodispersity

  • Limited proteolysis to confirm compact folding

• Optimized crystallization conditions: For X-ray crystallography, screening with membrane-mimetic environments such as bicelles, nanodiscs, or lipidic cubic phases can improve crystal formation of the membrane-associated KdpC.

• Alternative structural approaches: Cryo-electron microscopy has proven particularly successful for membrane protein complexes and may be preferable to crystallography for the complete Kdp system.

Successful structural studies have typically employed fusion protein approaches (such as T4 lysozyme insertions or BRIL fusions) to enhance crystallization propensity, or have relied on antibody fragments to stabilize specific conformations of the protein.

How do recent findings on c-di-AMP signaling inform new approaches to studying KdpC function in B. anthracis?

Recent discoveries regarding c-di-AMP signaling provide new avenues for investigating KdpC function:

• Regulatory mechanisms: The finding that c-di-AMP inhibits Kdp operon expression through binding to KdpD and the ydaO riboswitch suggests a complex regulatory network controlling potassium transport . Researchers can now design experiments to manipulate c-di-AMP levels using phosphodiesterase inhibitors or genetic approaches to modulate KdpC expression and study functional consequences.

• Connection to virulence: The link between c-di-AMP, potassium uptake, and anthrax toxin expression provides a mechanistic framework for understanding how environmental signals are integrated into virulence regulation . This connection can be exploited to develop novel anti-virulence strategies targeting this signaling network.

• Methodological advances: New biosensors for c-di-AMP based on fluorescence or BRET allow real-time monitoring of signaling dynamics in living cells. These tools can be combined with potassium-specific probes to correlate c-di-AMP fluctuations with potassium transport activity and virulence factor expression.

• Structural insights: Cryo-EM structures of c-di-AMP-bound KdpD have revealed conformational changes that propagate to affect interactions with KdpE and ultimately KdpC expression. These structural details allow for rational design of mutations to disrupt or enhance these regulatory interactions.

Future research should focus on defining the precise molecular mechanisms by which potassium levels influence anthrax toxin expression, potentially revealing new targets for therapeutic intervention.

What potential does the KdpC protein hold as a target for developing novel antimicrobial approaches against B. anthracis?

The KdpC protein represents a promising but underexplored target for novel antimicrobial strategies against B. anthracis for several reasons:

• Essentiality under relevant conditions: While not absolutely essential under laboratory conditions, the Kdp system becomes critical during host infection when potassium is limited. Inhibiting KdpC function could therefore impair bacterial survival during infection.

• Impact on virulence: The connection between potassium homeostasis and toxin expression suggests that targeting KdpC could reduce virulence without directly killing the bacteria, potentially reducing selective pressure for resistance development .

• Structural uniqueness: The Kdp-ATPase complex has unique structural features compared to eukaryotic transporters, offering opportunities for selective targeting.

• Targeting strategies:

  • Small molecule inhibitors that disrupt KdpC-KdpB interactions

  • Peptide mimetics that compete for binding sites within the complex

  • Allosteric modulators that lock the transport complex in inactive conformations

  • Anti-sense RNA or CRISPR-based approaches to reduce KdpC expression

• Combination approaches: Targeting KdpC alongside conventional antibiotics may increase efficacy, particularly in environments where potassium is limited.

Preliminary screening efforts have identified several compounds that selectively inhibit the Kdp-ATPase complex in vitro, with IC₅₀ values in the micromolar range. These compounds demonstrate synergistic effects when combined with β-lactam antibiotics against B. anthracis in potassium-limited conditions, highlighting the therapeutic potential of this approach.

How does the KdpC protein interact with the B. anthracis S-layer and capsule formation pathways?

The relationship between KdpC and B. anthracis surface structures remains an emerging area of research:

• S-layer interactions: While direct interactions between KdpC and S-layer proteins (Sap and EA1) have not been definitively established, potassium homeostasis may affect the secretion and assembly of these structures . The SecA2 and SlaP proteins, which facilitate S-layer protein secretion, may be functionally influenced by potassium levels regulated by the Kdp system.

• Capsule regulation: The poly-γ-D-glutamate capsule, a critical virulence factor of B. anthracis that helps it evade the host immune response, requires proper ion homeostasis for optimal synthesis . Preliminary data suggest that disruptions in potassium transport affect capsule production, though the precise mechanisms remain to be elucidated.

• Experimental approaches: Researchers investigating these connections should consider:

  • Co-immunoprecipitation and pull-down assays to identify direct protein-protein interactions

  • Fluorescence microscopy with labeled proteins to visualize potential co-localization

  • Transcriptomics to identify coordinated expression patterns

  • Electron microscopy to assess changes in surface structures in kdpC mutants

Studies using super-resolution microscopy have suggested that components of the Kdp system may localize non-uniformly in the bacterial membrane, with potential enrichment near areas of active S-layer assembly. This spatial organization could facilitate local ion homeostasis required for proper assembly of surface structures.

How does the KdpC protein from B. anthracis compare with homologs from other bacterial pathogens?

Comparative analysis of KdpC across bacterial species reveals important evolutionary and functional insights:

Key findings from comparative studies include:

• Conserved core domains: A central core region (residues 45-120) is highly conserved across species, suggesting essential structural or functional importance.

• Differential regulation: While the basic function in potassium transport is conserved, regulatory mechanisms vary. For example, the c-di-AMP regulatory pathway appears more prominent in Gram-positive bacteria like B. anthracis compared to Gram-negative species.

• Evolutionary adaptation: Variations in KdpC sequences correlate with the ecological niches of different bacteria, with soil-dwelling species showing adaptations for fluctuating potassium availability.

• Host interaction: Pathogen-specific adaptations in KdpC may reflect requirements for survival during host infection, including responses to host-imposed potassium limitation as an antimicrobial strategy.

Researchers can exploit these differences to develop species-specific inhibitors or to better understand the evolution of potassium transport systems in bacterial pathogens.

What methodological differences should be considered when studying KdpC in B. anthracis compared to model organisms?

When transitioning from model organisms to B. anthracis, researchers must adapt their experimental approaches to account for several important differences:

• Biosafety considerations: B. anthracis is a BSL-3 pathogen, requiring appropriate containment facilities and protocols not needed for model organisms . Researchers often employ attenuated strains (such as the Sterne strain lacking the pXO2 plasmid) for initial studies before confirming key findings in fully virulent strains.

• Genetic manipulation: Transformation efficiencies are typically lower in B. anthracis compared to model organisms like E. coli or B. subtilis. Methods such as electroporation require optimization, with typical protocols using higher field strengths (2-2.5 kV/cm) and specialized buffer compositions.

• Growth conditions: B. anthracis has specific media requirements and growth patterns that differ from model organisms. For Kdp studies, chemically defined media with controlled potassium concentrations are essential, but must be appropriately adapted for B. anthracis physiology.

• Protein expression systems: Heterologous expression of B. anthracis proteins in E. coli may face challenges including codon bias, misfolding, or toxicity. Codon-optimized sequences and specialized expression strains can help overcome these issues.

• Virulence context: Unlike model organisms, studies of KdpC in B. anthracis should consider its relationship to virulence factors. This requires additional assays for toxin production and capsule formation that aren't relevant in non-pathogenic models .

Researchers have successfully adapted techniques from model organisms by developing B. anthracis-specific protocols, including optimized electroporation methods that increase transformation efficiency by up to 100-fold and specialized expression vectors with promoters that function robustly in the B. anthracis cellular environment.

How might systems biology approaches enhance our understanding of KdpC function in the context of B. anthracis physiology?

Systems biology offers powerful approaches to contextualize KdpC within the broader cellular network:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data from wild-type and kdpC mutant strains under various conditions can reveal:

  • Global regulatory networks affected by potassium transport

  • Metabolic adaptations to potassium limitation

  • Unexpected functional connections to other cellular processes

• Computational modeling: Mathematical models of ion transport, metabolism, and gene regulation can predict:

  • The impact of KdpC function on cellular energetics

  • Threshold effects in potassium-dependent cellular processes

  • System-level responses to perturbations in potassium homeostasis

• Protein interaction networks: High-throughput protein-protein interaction studies using techniques like BioID or APEX proximity labeling can map the protein neighborhood of KdpC, potentially revealing unexpected interaction partners.

• Single-cell approaches: Microfluidics-based single-cell analysis can reveal heterogeneity in potassium transport activity and its correlation with virulence factor expression at the individual cell level.

These approaches have already yielded insights into unexpected connections between potassium homeostasis and central carbon metabolism in B. anthracis, with evidence suggesting that changes in potassium levels influence glycolytic enzyme activity through allosteric mechanisms. Such integrated approaches promise to reveal how KdpC function is coordinated with broader cellular physiology to optimize bacterial survival and virulence.

What is the potential for structural vaccinology approaches targeting KdpC for anthrax prevention?

Structural vaccinology targeting KdpC represents an innovative approach to anthrax prevention, though with specific challenges: